Reversible protein phosphorylation is a fundamental posttranslational control mechanism in eukaryotes. Protein phosphatase 2A (PP2A) is one of the major classes of Ser/Thr phosphatases with likely thousands of phospho-protein substrates. The predominant form of PP2A is a heterotrimer of catalytic, scaffolding, and variable regulatory subunits. The identity of the regulatory subunit defines substrate specificity, subcellular localization, and regulation of PP2A by second messengers and reversible phosphorylation. The recent crystal structure of one of the several dozen possible PP2A heterotrimers offered a first glimpse into the molecular architecture and regulation of this fascinating enzyme. Still, substrates and physiological functions of specific PP2A heterotrimers remain poorly defined. Here, we propose to characterize two brain-specific PP2A regulatory subunits with relevance to neurodegenerative disorders, B'? and B?2. B'? acts as a gatekeeper in the regulation of tyrosine hydroxylase (TH) activity and catecholamine synthesis. Inhibitors of PP2A enzymes containing this subunit may therefore prove useful for the treatment of Parkinson's disease (PD). B?2 is derived from a gene mutated in spinocerebellar ataxia type 12 (SCA12). B?2 expression is pro-apoptotic, while B'? silencing protects neurons against a variety of insults. PP2A/B?2 inhibitors may thus form the basis for novel neuroprotective therapies. The first aim of this application is exploratory in nature, while the remaining two are hypothesis-driven. Aim 1 uses quantitative proteomics to identify interactors and substrates of PP2A/B'? and PP2A/B?2 in stable PC12 cell lines that inducibly express or silence these neuronal regulatory subunits. Aims 2 and 3 investigate how these two neuron-specific PP2A holoenzymes are regulated and how they mediate disease-related substrate dephosphorylation in vitro, in cell culture, and in animal models. Based on recent PP2A hetereotrimer crystal structures, aim 2 examines structural determinants of TH dephosphorylation by B'?, regulation of PP2A/B'? by kinases, and regulation of dopamine synthesis by B'? in vivo. Lastly, aim 3 seeks to define the mechanisms by which B?2 promotes apoptosis. We hypothesize that PP2A/B?2 dephosphorylates and inactivates mitofusin-2, a protein mutated in a common peripheral neuropathy, Charcot-Marie-Tooth disease type 2A. These studies are significant in that they advance our understanding of the critical regulatory enzyme PP2A with the ultimate goal of better therapies for several neurodegenerative disorders.